WO2021233314A1 - METHODS FOR PREPARING β-D-(1,4)-MANNURONATE OLIGOSACCHARIDE AND INTERMEDIATE THEREOF - Google Patents

Abstract

Provided is a method for economically and efficiently preparing β-D-(1,4)-mannuronate oligosaccharide as represented by formula (IX). Particularly, the method comprises: using economical and easily available 1,2,3,4,6-penta-O-acetyl-D-mannopyranose (formula X) as a raw material to prepare a key intermediate compound (I), compound (II), and compound (III); coupling the intermediate compounds (II) and (III) into an oligosaccharide receptor compound (V), and coupling the intermediate compounds (I) and (II) into an oligosaccharide donor compound (VII); and coupling the compound (V) and the compound (VII) into an oligosaccharide compound (VIII), and performing protecting group removal on the oligosaccharide compound (VIII) to obtain β-D-(1,4)-mannuronate oligosaccharide (IX).

Description

Preparation method of β-D-(1,4)-mannuronic acid oligosaccharide and its intermediate Technical field

The invention belongs to the field of organic chemistry synthesis, and relates to a preparation method of β-D-(1,4)-mannuronic acid oligosaccharides (disaccharides to eicosanose).

Background technique

β-D-(1,4)-mannuronic acid oligosaccharides are widely present in natural products. They can promote the growth of plant roots, inhibit bacteria, and promote the production of human keratinocytes. It can interact with Toll- like receptors 2 and 4 Combined, it shows the effect of immunomodulation (CN103275133A), β-D-(1,4)-mannuronic acid oligosaccharide also has a good effect in the treatment of vascular dementia (CN106344593A). On November 2, 2019, the National Food and Drug Administration approved the listing of Manrutner Capsules for the treatment of mild to moderate Alzheimer’s disease, the main ingredient of which is β-D-(1,4)-mannuronic acid Oligosaccharides and their derivatives.

At present, β-D-(1,4)-mannuronic acid oligosaccharides are mainly obtained by degrading sodium alginate (CN100508985C). The degradation preparation process presents greater challenges to its purity and impurity control. There is an urgent need to develop an efficient, Simple operation and precise control of the preparation process. There are reports in the literature using solid-phase synthesis (WO2012138698) or liquid-phase synthesis (Codée, Jeroen DC, van den Bos, Leendert J, de Jong, Ana-Rae, et al. The Stereodirecting Effect of the Glycosyl C5-Carboxylate Ester: Stereoselective Synthesis of β-Mannuronic Acid Alginates[J].Journal of Organic Chemistry,74(1):38-47) Preparation of β-D-(1,4)-mannuronic acid oligosaccharide derivatives, in which the solid phase The synthesis method has high cost, high equipment requirements, and is not easy to industrialize production. In addition, the existing liquid-phase synthesis method has problems such as low synthesis efficiency and complicated removal of protective groups, which is not easy for industrial production. Therefore, the development and research of a new, efficient and process-controllable preparation method of β-D-(1,4)-mannuronic acid oligosaccharide has important application value and economic value.

Summary of the invention

The first aspect of the present invention provides a method for preparing β-D-(1,4)-mannuronic acid oligosaccharides, which can economically and efficiently prepare high-purity β-D-(1,4)- Mannouronic acid oligosaccharides. The method includes coupling compound V and compound VII and selectively removing the protecting group R ^{3 to} generate compound VIII;

And optionally, removing the protecting group R ^{2 of} compound VIII at one time to generate β-D-(1,4)-mannuronic acid oligosaccharide as shown in formula IX;

Wherein, m is selected from an integer of 2-18; n and n'are each independently selected from an integer of 0-8; R ^{1 is} selected from C _1-8 alkyl, optionally substituted _{by C 1-8 alkyl 6-14} aryl group; R ² is a hydroxyl protecting group that can be removed by hydrogenation reaction catalyzed by palladium-carbon or oxidation reaction catalyzed by palladium-carbon ^{; R 3 is hydrogenation reaction catalyzed by palladium-carbon or oxidation reaction catalyzed by palladium-carbon} Removed hydroxyl protecting group.

In particular, the present invention can realize the preparation of intermediate compounds I, II, and 1,2,3,4,6-Penta-O-acetyl-D-mannanose (formula X), which are economically and easily available III; Then the intermediate compounds II and III are coupled to form an oligosaccharide acceptor compound V, and the intermediate compounds I and II are coupled to form an oligosaccharide donor compound VII; then compound V and compound VII are coupled to form an oligosaccharide compound VIII, the protective group is removed to obtain the final product compound IX, which is β-D-(1,4)-mannuronic acid oligosaccharide. The present invention provides an economical and efficient solution for the synthesis of β-D-(1,4)-mannuronic acid oligosaccharide compounds with a degree of polymerization of 2 to 20, which is comparable to the process of extracting and preparing oligosaccharide mixtures such as degrading sodium alginate. In comparison, this scheme can obtain oligosaccharides with a single degree of polymerization and high purity, which lays a solid foundation for further research on the pharmacological and biological activities of oligosaccharides with a single degree of polymerization.

The second aspect of the present invention provides the key intermediate compound I, compound II, compound III, compound V, compound VII and compound VIII for the synthesis of β-D-(1,4)-mannuronic acid oligosaccharides, with structural formula As shown below:

in,

R ^{1 is} selected from C _1-8 alkyl, C _{6-14 aryl optionally substituted by C 1-8} alkyl; preferably, R ^{1 is} selected from phenyl, o-tolyl, p-methylphenyl, 4-tert-butyl-2-methylphenyl, 2,4-di-tert-butylphenyl, methyl or ethyl;

R ² is a hydroxyl protecting group that can be removed by a hydrogenation reaction catalyzed by palladium on carbon; preferably, R ^{2 is} selected from C _6-14 arylmethyl or allyl, and the C _6-14 arylmethyl optionally Is substituted by C _1-8 alkyl, C _1-8 alkoxy, halogen; more preferably, R ^{2 is} selected from benzyl, p-methoxybenzyl, naphthylmethyl, and allyl; most preferably, R ^{2 is} selected from benzyl;

R ³ is a hydroxyl protecting group that cannot be removed by hydrogenation reaction catalyzed by palladium-carbon; preferably, R ^{3 is} selected from C _1-8 alkyl acyl, C _1-8 alkoxy acyl, C _6-14 aryl acyl , Tris (C _1-8 alkyl) silyl, 9-fluorenyl methoxy formyl, tris (C _6-14 aryl) methyl; wherein C _1-8 alkyl acyl and C _1-8 alkane Any carbon atom in the C _1-8 alkyl group in the oxyacyl group may be optionally oxo; more preferably, R ^{3 is} selected from the group consisting of acetyl, levulinyl, trimethylsilyl, tert-butyl di Methylsilyl, benzoyl, 9-fluorenylmethoxyformyl, or trityl; most preferably, R ^{3 is} selected from levulinyl;

R ^{4 is} selected from H, C _1-8 alkyl, C _6-14 aryl;

X is selected from fluorine, chlorine, bromine, and iodine;

m is selected from an integer of 2-18; preferably, m is selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18;

n and n'are each independently selected from an integer of 0-8; preferably, n and n'are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, or 8.

Description of the drawings

Figure 1 shows the general synthetic route of compound VIII and compound IX.

Figure 2 shows the synthetic route of compound IX-1 and compound IX-2.

Figure 3 shows the synthetic route of compound IX-3 and compound IX-4.

Figure 4 shows the synthetic route of compound IX-5.

Detailed ways

Unless otherwise specified, the term "alkyl" mentioned herein can be a linear or branched saturated hydrocarbon group, such as methyl, ethyl, propyl, butyl, octyl, isopropyl, tert-butyl, Sec-pentyl and similar groups. The alkyl group may be unsubstituted or substituted with one or more substituents (for example, halogen, alkoxy, aryl, aralkyl, aralkoxy, and the like). C _1-n alkyl (where n is an integer) refers to an alkyl group containing 1-n carbon atoms, for example, 1-18 carbon atoms, 1-12 carbon atoms, 1-10 carbon atoms, 1-8 Carbon atoms, 1-6 carbon atoms, 1-4 carbon atoms, etc.

The term "aryl" refers to a monovalent unsaturated aromatic group having a single ring (such as phenyl) or a condensed ring (such as naphthyl or anthracenyl), optionally halogenated (including fluorine, chlorine, bromine, iodine) , Alkyl, aralkyl, alkoxy, aralkoxy and similar substituents are substituted. The C _6-n aryl group (wherein n is an integer) refers to an aryl group having 6 to n carbon atoms, such as phenyl, naphthyl, anthryl, or an optionally substituted group thereof.

The first aspect of the present invention relates to a method for preparing β-D-(1,4)-mannuronic acid oligosaccharides represented by formula (VIII) or formula (IX),

include:

Compound V and compound VII are subjected to a coupling reaction and the protective group R ^{3 is} selectively removed to generate compound VIII;

And optionally, removing the protecting group R ^{2 of} compound VIII at one time to generate β-D-(1,4)-mannuronic acid oligosaccharide as shown in formula IX;

Wherein, m is selected from an integer of 2-18; n and n'are each independently selected from an integer of 0-8; R ^{1 is} selected from C _1-8 alkyl, optionally substituted _{by C 1-8 alkyl 6-14} aryl group; R ² is a hydroxy protecting group that can be removed by hydrogenation catalyzed by palladium on carbon; R ³ is a hydroxy protecting group that cannot be removed by hydrogenation catalyzed by palladium on carbon.

The above coupling reaction is carried out in the presence of a large sterically hindered organic base, diphenyl sulfoxide and sulfonic anhydride catalyst; and then the hydroxyl protecting group R ^{3 at the} 4-position and the hydroxyl protecting group R ^{2 at} other positions are successively removed to obtain as shown in IX The β-D-(1,4)-mannuronic acid oligosaccharide compound shown. Preferably, the sulfonic anhydride catalyst is selected from methanesulfonic anhydride, trifluoromethanesulfonic anhydride or p-toluenesulfonic anhydride.

In one embodiment of the present invention, a method for synthesizing compound V is provided, including:

Dissolve Intermediate Compound II and Intermediate Compound III in a suitable organic solvent, add dry molecular sieves, protected by nitrogen or argon, add a sulfonic acid catalyst at a suitable temperature, and generate 1,4-glycosidic bonds in the coupling reaction. Selectively remove the 4-position hydroxyl protecting group to obtain compound IV; repeat the coupling reaction of compound IV and compound II and the steps of selectively removing the 4-position hydroxyl protecting group R ³ , and optionally continue the obtained compound Repeat the steps of coupling reaction and deprotection until compound V is obtained.

In a preferred embodiment of the present invention, the organic solvent in the synthesis of compound V is selected from: anhydrous dichloromethane, anhydrous tetrahydrofuran, anhydrous ether, anhydrous N,N-dimethylformamide, anhydrous N,N-dimethylacetamide, anhydrous toluene or anhydrous dimethylsulfoxide, preferably anhydrous dichloromethane; the sulfonic acid catalyst is selected from: methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid Or trimethylsilyl trifluoromethanesulfonate, preferably trimethylsilyl trifluoromethanesulfonate; the reagent for removing the 4-position hydroxyl protecting group is preferably hydrazine acetate, and the molar equivalent of hydrazine acetate is 3-8eq , Preferably 3-5.5eq, for example 4.3eq.

In one embodiment of the present invention, a method for synthesizing compound VII is provided, including:

Dissolve compound I and compound II in a suitable organic solvent, add dry molecular sieve, nitrogen or argon protection, add a sulfonic acid catalyst at a suitable temperature, the coupling reaction generates 1,4-glycosidic bonds, and then selectively remove Remove the 4-position hydroxy protecting group R ³ to obtain compound VI; repeat the above coupling reaction and selectively remove the 4-position protecting group R ³ steps of compound VI and compound II; optionally continue repeating the obtained compound The steps of coupling reaction and deprotection are mentioned above until compound VII is obtained.

In a preferred embodiment of the present invention, the organic solvent in the synthesis of compound VII is selected from: anhydrous dichloromethane, anhydrous tetrahydrofuran, anhydrous ether, anhydrous N,N-dimethylformamide, anhydrous N,N-dimethylacetamide, anhydrous toluene or anhydrous dimethylsulfoxide, preferably anhydrous dichloromethane; the sulfonic acid catalyst is selected from: methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid Or trimethylsilyl trifluoromethanesulfonate; the sulfonic acid catalyst is preferably trimethylsilyl trifluoromethanesulfonate; the reagent for removing the 4-position hydroxyl protecting group is preferably hydrazine acetate.

In one embodiment of the present invention, a method for synthesizing compound IX is provided, including:

Dissolve compound V and compound VII in a suitable organic solvent, add dry molecular sieve, protected by nitrogen or argon, at a suitable temperature, add large hindered organic base, diphenyl sulfoxide and sulfonic anhydride to catalyze the coupling reaction Generate 1,4-glycosidic bonds, and then selectively remove the protective group R ^{3 of the} 4-position hydroxyl group to obtain compound VIII; add palladium on carbon (palladium content 5%-10%) to compound VIII, and remove all R from compound VIII by hydrogenation ² protecting groups to obtain β-D-(1,4)-mannuronic acid oligosaccharide compound IX.

In a preferred embodiment of the present invention, the molar ratio of compound V and compound VII in the synthesis of compound IX is 1:0.9; the solvent for the coupling reaction is selected from: anhydrous dichloromethane, anhydrous tetrahydrofuran, Anhydrous ether, anhydrous N,N-dimethylformamide, anhydrous N,N-dimethylacetamide, anhydrous toluene or anhydrous dimethyl sulfoxide, preferably anhydrous dichloromethane; The hindered organic base is selected from: 1,8-diazabicycloundec-7-ene, 2,6-di-tert-butylpyridine or 2,4,6-tri-tert-butylpyrimidine, preferably 2,6- Di-tert-butylpyridine; the sulfonic anhydride catalyst is selected from: methanesulfonic anhydride, trifluoromethanesulfonic anhydride or p-toluenesulfonic anhydride, preferably trifluoromethanesulfonic anhydride; the molar equivalent of sulfonic anhydride is preferably 0.05eq; the reaction temperature is preferably -60°C; the reagent used to remove the protecting group of the 4-hydroxyl group is preferably hydrazine acetate, and its molar equivalent is preferably 4.3 eq; the temperature for removing the protecting group is preferably 25°C.

The starting compounds of Formula I and Formula II used above can be obtained from the compound of Formula X, respectively.

The compound of formula X is 1-5 substituted mannanose, wherein the substituent may be a C ₁ -C ₆ acyl group, such as formyl, acetyl, and propionyl. An example of a compound of formula X is 1,2,3,4,6-penta-O-acetyl-D-mannanose.

In a preferred embodiment of the present invention, 1,2,3,4,6-penta-O-acetyl-D-mannanose (compound represented by formula X), which is a simple and easily available raw material, is taken as an example, The overall reaction route is as follows:

Wherein R ⁵ is a C _1-8 acyl group, preferably an acetyl group.

In one embodiment of the present invention, a method for synthesizing intermediate compound I is provided, including:

Step 1: 1,2,3,4,6-penta-O-acetyl-D-mannanose ^{reacts with the anomeric carbon protective agent R 1} SH, and then performs an alkaline hydrolysis reaction to obtain compound A;

Step 2: Selectively protect the 2 and 3 hydroxyl groups of compound A to obtain compound B;

Step 3: Oxidize the hydroxyl group at the 6-position of compound B to a carboxyl group, and then esterify the carboxyl group to obtain compound I.

In a preferred embodiment, the method includes:

Step 1. In a dry organic solvent, using 1,2,3,4,6-penta-O-acetyl-D-mannanose as a raw material, stirring at an appropriate temperature in the presence of an acidic catalyst, Protected by nitrogen or argon, add a suitable anomeric carbon protective agent R ¹ SH for reaction, and then conduct alkaline hydrolysis to obtain compound A;

Step 2. Under the catalysis of Lewis acid, select an appropriate hydroxyl protecting group to selectively protect the 4 and 6 hydroxyl groups of compound A, then selectively protect the 2 and 3 hydroxyl groups, and finally selectively remove them Protecting groups at positions 4 and 6 give compound B;

Step 3. In an organic solvent, add an oxidizing agent to compound B to selectively oxidize the 6-position hydroxyl group of compound B to a carboxyl group, and then react with an alkylating agent to form an ester under alkaline conditions at an appropriate temperature and solvent. Compound I is obtained.

In a more preferred embodiment, the synthesis of the intermediate compound I adopts a one-pot two-step reaction, and the intermediate does not need to be separated and purified. Wherein, the organic solvent in step 1 is selected from: anhydrous dichloromethane, anhydrous tetrahydrofuran, anhydrous toluene, anhydrous N,N-dimethylformamide or anhydrous N,N-dimethylacetamide; The appropriate temperature is 0-25°C, preferably 0-5°C; the acid catalyst is selected from: boron trifluoride ether, acetyl chloride or hydrogen chloride gas, preferably boron trifluoride ether; moles of boron trifluoride ether The equivalent is preferably 0.1eq; the base is selected from: sodium ethoxide, potassium ethoxide, magnesium ethoxide, sodium methoxide, potassium methoxide or magnesium methoxide, preferably sodium methoxide; the molar equivalent of sodium methoxide is preferably 0.1eq; the solvent for the alkali hydrolysis Selected from: methanol, ethanol or tetrahydrofuran, preferably methanol; the anomeric carbon protective agent added in the reaction is selected from: thiophenol, o-toluenethiol, p-methylthiophenol, 4-tert-butyl-2- Methyl thiophenol, 2,4-di-tert-butyl thiophenol, methyl mercaptan or ethyl mercaptan, preferably p-methyl thiophenol; the molar equivalent of the anomeric carbon protecting agent is preferably 1.05 eq.

Wherein, the protecting agent used for protecting the 4-position and 6-position of compound A in step 2 is selected from: phthalic acetal, p-methoxy phthalic acetal or 2,2-dimethoxypropane (acetone Fork), preferably phthalic acetal; the molar equivalent of phthalic acetal is preferably 0.9eq; the Lewis acid catalyst is selected from: p-toluenesulfonic acid, trifluoromethanesulfonic acid, aluminum trichloride and trichloride Iron, preferably iron trichloride; the protective agent used to protect the 2-position and 3-position hydroxyl groups of compound A is selected from the group consisting of benzyl bromide, benzyl chloride, p-methoxybenzyl bromide, p-methoxybenzyl chloride or allyl Base bromide, preferably benzyl bromide; the equivalent of benzyl bromide is preferably 1.9 eq.

Wherein, the oxidant in step 3 is selected from: tetramethylpiperidine nitroxide, sodium hypochlorite, potassium bromide, hydrogen peroxide, tert-butyl hydroperoxide or iodobenzene diacetate, preferably tetramethylpiperidine nitroxide or Iodobenzene diacetate; the molar equivalent of tetramethylpiperidine nitrogen oxide is preferably 0.2eq, or the molar equivalent of iodobenzene diacetate is preferably 2.5eq; the organic solvent is selected from: dichloromethane, acetonitrile, water, tetrahydrofuran , N,N-dimethylformamide or any one or more of N,N-dimethylacetamide, preferably selected from: acetonitrile-water mixed solvent (the volume ratio of acetonitrile to water is about 5:1 -1:5), tetrahydrofuran-water mixed solvent (the volume ratio of tetrahydrofuran to water is about 5:1 to 1:5) or dichloromethane-water mixed solvent (the volume ratio of dichloromethane to water is about 5:1 -1:5), more preferably a dichloromethane-water mixed solvent (the volume ratio of dichloromethane to water is about 1:2); the temperature of the oxidation reaction is 0°C to 25°C, and the preferred temperature is 25°C The base is selected from: potassium carbonate, cesium carbonate, sodium carbonate, calcium carbonate, silver carbonate, triethylamine or diisopropylethylamine, preferably potassium carbonate or triethylamine; the molar equivalent of the base is preferably 0.5eq to 3eq, more preferably 1.5eq of potassium carbonate or 1eq of triethylamine; the alkylating reagent is selected from: benzyl bromide, benzyl chloride, p-methoxybenzyl bromide, and p-methoxybenzyl chloride Or allyl bromide, preferably benzyl bromide; the solvent for the alkylation reaction is selected from: tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, acetone or acetonitrile, preferably acetone; The temperature of the alkylation reaction is 25°C to 100°C, and the preferred temperature is 30°C.

In one embodiment of the present invention, a method for synthesizing intermediate compound II is provided, including:

Step 1: Protect the 4-hydroxyl of compound I to obtain compound D;

Step 2: Selectively remove the anomeric carbon protecting group -SR ^{1 of} compound D to obtain compound E;

Step 3: Under basic conditions, compound E is reacted with CX ₃ CN or CX ₃ C(=NR ⁴ )X to obtain compound II.

In a preferred embodiment, the method includes:

Step 1: In a dry organic solvent, under alkaline conditions, use a suitable protective agent to protect the 4-hydroxyl of compound I to obtain compound D;

Step 2: Selectively remove the anomeric carbon protecting group -SR ^{1 of} compound D to obtain compound E;

Step 3: In a dry organic solvent, under alkaline conditions, compound E is reacted with CX ₃ CN or CX ₃ C(=NR ⁴ )X (for example, trichloroacetonitrile) to obtain compound II.

In a more preferred embodiment, the synthesis of the intermediate compound II adopts a one-pot three-step reaction, and the intermediate does not need to be separated and purified. Wherein, the suitable protective agent in step 1 is selected from acetyl chloride, acetic anhydride, trimethylchlorosilane, tert-butyldimethylchlorosilane, benzoyl chloride, 9-fluorenylmethyl chloroformate, triphenyl Methyl chloride, levulinyl chloride or levulinic acid, preferably levulinic acid; the molar equivalent of the protective agent is preferably 1.5eq; the organic solvent is selected from: anhydrous dichloromethane, anhydrous tetrahydrofuran, anhydrous ethyl acetate, Anhydrous acetonitrile, anhydrous toluene, anhydrous N,N-dimethylformamide or anhydrous N,N-dimethylacetamide, preferably anhydrous dichloromethane; the preferred reaction temperature is 25°C; the alkali Selected from: anhydrous potassium carbonate, anhydrous sodium carbonate, triethylamine, N,N-p-dimethylaminopyridine or pyridine, preferably N,N-p-dimethylaminopyridine; the molar equivalent of the base is preferably 1.5eq .

Wherein, the reagent for removing the protective group of the anomeric carbon in step 2 is selected from N-chlorosuccinimide or N-bromosuccinimide, preferably N-bromosuccinimide ; The reaction temperature is preferably 25°C.

Wherein, the organic solvent in step 3 is selected from: anhydrous dichloromethane, anhydrous tetrahydrofuran, anhydrous ethyl acetate, anhydrous acetonitrile, anhydrous toluene, anhydrous N,N-dimethylformamide or anhydrous N,N-dimethylacetamide, preferably anhydrous dichloromethane; the reaction temperature is preferably 0°C to 10°C; the base is selected from: anhydrous potassium carbonate, anhydrous sodium carbonate, triethylamine, 1,8 -Diazabicyclo[5.4.0]undec-7-ene, N,N-p-dimethylaminopyridine or pyridine, preferably 1,8-diazabicyclo[5.4.0]undec-7 -En; The molar equivalent of the base is preferably 0.5 eq.

In one embodiment of the present invention, a method for synthesizing intermediate compound III is provided, including:

Step 1: Protect the 4-hydroxyl of compound I to obtain compound D;

Step 2: Under alkaline conditions, ^{react compound D with R 2} OH to obtain compound F;

^{Step 3: Selectively remove the protective group R 3} of the 4-hydroxyl group of compound F to obtain compound III.

In a preferred embodiment, the method includes:

Step 1. In a dry organic solvent, under alkaline conditions, use a suitable protective agent to protect the 4-position hydroxyl group of compound I to obtain compound D;

Step 2. In a dry organic solvent, in the presence of a large hindered base, R ² OH, diphenylthiophenol and sulfonic anhydride catalyst are added to compound D to react to obtain compound F;

Step 3. Selectively remove the protecting group of the 4-hydroxyl group of compound F in an organic solvent to obtain compound III.

In a more preferred embodiment of the present invention, the synthesis of intermediate compound III adopts a one-pot three-step method. Wherein, the suitable protective agent in step 1 is selected from: acetyl chloride, acetic anhydride, trimethylchlorosilane, tert-butyldimethylchlorosilane, 9-fluorenylmethyl chloroformate, benzoyl chloride, trimethylchlorosilane, Phenyl chloride, levulinyl chloride or levulinic acid, preferably levulinic acid; the molar equivalent of the protective agent is preferably 1.5eq; the organic solvent is selected from: anhydrous dichloromethane, anhydrous tetrahydrofuran, anhydrous ethyl acetate, Anhydrous acetonitrile, anhydrous toluene, anhydrous N,N-dimethylformamide or anhydrous N,N-dimethylacetamide, preferably anhydrous dichloromethane; the reaction temperature is preferably 25°C; the alkali Selected from: anhydrous potassium carbonate, anhydrous sodium carbonate, triethylamine, N,N-p-dimethylaminopyridine or pyridine, preferably N,N-p-dimethylaminopyridine; the molar equivalent of the base is preferably 1.5eq .

Wherein, the organic solvent in step 2 is selected from: anhydrous dichloromethane, anhydrous tetrahydrofuran, anhydrous ether, anhydrous N,N-dimethylformamide, anhydrous N,N-dimethylacetamide, Anhydrous toluene or anhydrous dimethyl sulfoxide, preferably anhydrous dichloromethane; the large sterically hindered organic base is selected from: 1,8-diazabicycloundec-7-ene, 2,6-di Tert-butylpyridine or 2,4,6-tri-tert-butylpyrimidine, preferably 2,6-di-tert-butylpyridine; the molar equivalent of the base is preferably 2.2eq; the sulfonic acid anhydride catalyst is selected from: methanesulfonic anhydride, three Chloromethanesulfonic anhydride, tribromomethanesulfonic anhydride, trifluoromethanesulfonic anhydride or toluenesulfonic anhydride is preferably trifluoromethanesulfonic anhydride; the molar equivalent of trifluoromethanesulfonic anhydride is preferably 0.05eq; and the reaction temperature is preferably -60°C.

Wherein, the organic solvent in step 3 is dichloromethane and/or pyridine, preferably dichloromethane: pyridine=5:1 (V/V); it is used to selectively remove the 4-hydroxy protecting group of compound F The reagent is preferably hydrazine acetate, and its equivalent is preferably 4.3 eq.

In a preferred embodiment of the present invention, the compound I, compound II and compound III are respectively compounds I-1, II-1 and III-1 represented by the following structural formulas:

The advantages of the present invention are:

The method for preparing β-D-(1,4)-mannuronic acid oligosaccharides (Compound IX) described herein adopts a convergent synthesis strategy. Through the new oligosaccharide intermediates Compound I, Compound II and Compound III, The oligosaccharide donor (Compound VII) and the oligosaccharide acceptor (Compound V) are assembled into the oligosaccharide compound VIII with a higher number of sugar bases, and the protective group R ² in compound VIII is removed at one time to synthesize β-D-(1 , 4)-Mannouronic acid oligosaccharide (Compound IX). Compared with the prior art, the preparation method of the present invention is more concise, economical and efficient.

Example

The present invention will be further explained below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples are usually in accordance with conventional conditions or in accordance with the conditions recommended by the manufacturer.

The following example section provides the disaccharides (compound IX-1), tetrasaccharides (compound IX-2), trisaccharides (compound IX-3), pentasaccharides of β-D-(1,4)-mannuronic acid The total synthesis scheme of sugar (compound IX-4) and heptasaccharide (compound IX-5) further illustrates the present invention, but the present invention is not limited thereto.

Unless otherwise specified, the raw materials or reagents in the examples are all commercially available.

The room temperature in the examples refers to 20-30°C.

The normal pressure in the examples refers to 1 atmosphere.

The water in the examples refers to deionized water.

Meaning of acronyms

Abbreviations	meaning
TLC	TLC
EA	Ethyl acetate
Hex	Hexane
DCM	Dichloromethane
TEMPO	Tetramethylpiperidine Nitrogen Oxide
DIC	Diisopropylcarbodiimide
DMAP	Dimethylaminopyridine
NBS	Bromosuccinimide
DBU	1,8-diazabicycloundec-7- ene
TTBP	2,4,6-Tri-tert-butylpyridine
Tf 2 O	Trifluoromethanesulfonic anhydride
LevOH	Levulinic acid
TMSOTf	Trimethylsilyl Trifluoromethanesulfonate
CNCCl 3	Trichloroacetonitrile
min	minute
mL	Milliliters
mmol	Millimoles

Example 1: Synthesis of compound I-1

Step 1: Weigh 1,2,3,4,6-penta-O-acetyl-D-mannanose (19.5g, 50.0mmol) into a 500mL eggplant-shaped bottle, add anhydrous dichloride Methane (150 mL), the solution was colorless and transparent, in an ice bath, p-toluene thiophenol (6.2 g, 50.0 mmol) and boron trifluoride ether solution (12.7 mL, 100 mmol) were added. After stirring for half an hour in an ice bath, stirring was continued for 24 hours at room temperature, and the solution changed from light yellow to pink. TLC (EA/Hex=1/2) detects the disappearance of the raw materials, _{quenches the reaction with saturated NaHCO 3} solution, separates the dichloromethane layer with a separatory funnel, evaporates the solvent under reduced pressure, and then adds 200 mL of anhydrous methanol for reconstitution, and then adds methanol Sodium (256mg, 4.7mmol), stirred at room temperature for 12 hours, TLC (EA/Hex=1/2) detected that the raw material had reacted completely, adjusted the pH to neutral with dilute hydrochloric acid, evaporated the solvent under reduced pressure, and added dichloromethane (100mL ) And water (100 mL), stirred at room temperature for half an hour, separated the aqueous layer with a separatory funnel, evaporated under reduced pressure to remove the water to obtain the crude compound A-1 directly used in the next step without purification.

Step 2: Dissolve the crude compound A-1 (about 47.3mmol) in 200mL of anhydrous DMF, add PhCH(OMe) ₂ (7.9g, 52.03mmol, 1.1eq), p-toluenesulfonic acid (899mg, 4.7mmol, 0.1eq), the reaction temperature was controlled at 50°C, and the reaction was carried out under reduced pressure for 2 hours. TLC (CH ₃ OH/DCM=1/9) indicated that the reaction of the raw materials was complete. The reaction was cooled to room temperature, NaH (5.68g, 141.9mmol, 3.0eq) was added under an ice bath and reacted for 20 minutes, then BnBr (20.2g, 118.3mmol, 2.5eq) was added. After 20 minutes, the ice bath was removed and stirred at room temperature for 12 hours. , TLC (EA/Hex=1/4) indicates that the conversion of the raw material is complete. The NaH was quenched with methanol (10 mL) in an ice bath, the reaction solution was diluted by adding ethyl acetate (200 mL), and the reaction solution was washed three times with saturated brine (200 mL), concentrated under reduced pressure to remove the solvent, and then reconstituted by adding 300 mL of methanol. P-toluenesulfonic acid (899mg, 10%) was reacted at room temperature for 12 hours. TLC (EA/Hex=1/9) indicated that the reaction of the raw materials was complete. Then adjust the pH of the reaction solution to 8 with saturated sodium bicarbonate solution, separate the organic layer with a separatory funnel, remove the solvent under reduced pressure, and separate and purify by column chromatography. The eluent ratio (EA/Hex=1/12-CH ₃ OH/ DCM=1/9), spin-dry the organic phase, vacuum to constant weight, and obtain a white solid compound B-1 (11.8 g, 25.5 mmol) with a yield of 51% (yield based on acetylmannose). The NMR data is as follows:

¹ H NMR(600MHz, CDCl ₃ )δ7.37–7.21(m,12H), 7.06(d,J=7.9Hz,2H), 5.45(d,J=1.3Hz,1H), 4.60(d,J= 12.2Hz, 1H), 4.57–4.46(m, 3H), 4.17–4.00(m, 2H), 3.94(dd, J=3.0, 1.5Hz, 1H), 3.85–3.73(m, 2H), 3.68(dd ,J=9.1,3.1Hz,1H), 3.35(s,1H), 2.70(d,J=3.7Hz,1H), 2.28(s,3H).

The third step: Weigh compound B-1 (6.3g, 13.6mmol) into a 250mL eggplant-shaped bottle, add 87mL of a mixed solution of water and dichloromethane (v/v=1/2), which is colorless and transparent. Add TEMPO (424mg, 2.7mmol, 0.2eq), the solution turns reddish brown, then add PhI(OAc) ₂ (10.9g, 33.9mmol, 2.5eq), stir vigorously at room temperature for 3 hours, the solution turns brownish yellow, TLC (EA/Hex=1/1) indicates that the reaction of the raw materials is complete. Add a saturated Na ₂ S ₂ O ₃ aqueous solution (10 mL) to the reaction solution, adjust the pH to 3 with dilute hydrochloric acid, stir for 10 minutes, add 100 mL of dichloromethane for extraction, remove the solvent under reduced pressure, and then add 180 mL of acetone for reconstitution. Add BnBr (4.6g, 27.2mmol, 2eq), K ₂ CO ₃ (2.8g, 20.4mmol, 1.5eq), under nitrogen protection, stir the reaction at room temperature for 3 hours, the solution is reddish brown, TLC (CH ₃ OH/DCM =1/9) indicates that the raw materials have completely disappeared. The reaction solution was adjusted to neutral pH with dilute hydrochloric acid, and the solution was extracted and separated with dichloromethane (100mL). The organic phase was concentrated under reduced pressure and purified by column chromatography (EA/Hex=1/ 10-1/1) to obtain compound I-1 (4.3 g, 7.5 mmol) as a reddish-brown oil, with a yield of 55% (calculated as compound B-1). The NMR spectrum data is as follows:

¹ H NMR(600MHz, CDCl ₃ )δ7.70–7.28(m,17H), 6.97(d,J=7.9Hz,2H), 5.23(dd,J=44.0,12.3Hz,2H), 5.00(d, J = 11.4 Hz, 1H), 4.86 (d, J = 11.4 Hz, 1H), 4.75 (q, J = 12.0 Hz, 2H), 4.68 (d, J = 0.9 Hz, 1H), 4.45 (td, J = 9.5,2.5Hz,1H),4.10(t,J=6.4Hz,1H), 3.76(d,J=9.6Hz,1H),3.58–3.41(m,1H),3.08(d,J=2.5Hz, 1H), 2.30(s, 3H).

Example 2: Synthesis of compound II-1

The first step: Weigh compound I-1 (15.0g, 26.3mmol) into a 1000mL eggplant-shaped bottle, add 400mL of anhydrous dichloromethane, the solution is reddish brown, add LevOH (7.6g, 65.8mmol, 2.5eq ), DIC (8.3g, 10.2mL, 65.8mmol, 2.5eq), and DMAP (8.1g, 65.8mmol, 2.5eq), stirred at room temperature for 3 hours, the solution became brownish yellow and turbid. TLC (EA/Hex=1/2) indicates that the conversion of the raw material is complete. Saturated brine (500 mL) was added, the liquids were separated by extraction, and the organic phase was concentrated under reduced pressure to obtain the crude product compound D-1, which was directly used in the next step.

Step 2: Dissolve the crude compound D-1 in 320mL of acetone/water (v/v=15/1), add NBS (16.8g, 4eq) as a pale yellow solution, turn it into reddish brown after a few minutes, stir at room temperature for 1 Hours, the solution turns pale yellow. TLC (EA/Hex=1/2) indicates that the conversion of the raw material is complete. The reaction was quenched with saturated Na ₂ S ₂ O ₃ solution (10 mL), and dichloromethane (500 mL) was added for extraction. The organic layer was washed once with saturated brine, and concentrated under reduced pressure to remove the solvent to obtain a yellow oil compound E-1. Use directly In the next step.

Step 3: Dissolve the crude compound E-1 from the previous step in 90mL of anhydrous dichloromethane, add CNCCl ₃ (6.3g, 4.3mL, 5eq) and DBU (663mg, 0.7mL, 4.4mmol, 0.5eq), solution It was dark brown and stirred under ice bath for 3 hours. TLC (EA/Hex=1/2) indicates that the conversion of the raw material is complete. The reaction solution was concentrated under reduced pressure, and separated and purified by column chromatography. The eluent ratio (EA/Hex=1/6-1/1) gave compound II-1 (13.5g, 19.2mmol) as a pale yellow oil with a yield of 73% ( Based on compound I-1). The NMR data is as follows:

¹ H NMR (400MHz, CDCl ₃ ) δ7.43-7.16 (m, 15H), 6.53 (d, J = 3.3 Hz, 1H), 5.60 (t, J = 7.5 Hz, 1H), 5.12 (t, J = 12.9Hz, 1H), 5.03 (d, J = 12.2 Hz, 1H), 4.88 (d, J = 46.7 Hz, 2H), 4.65 (ddd, J = 34.3, 16.0, 6.9 Hz, 1H), 4.51 (d, J=7.6Hz,1H), 4.49–4.38(m,1H), 3.89(dd,J=7.8,2.9Hz,1H), 3.85–3.76(m,1H), 2.72–2.44(m,3H), 2.39 (d,J=7.2Hz,1H), 2.15(d,J=11.7Hz,3H).

Example 3: Synthesis of compound III-1

Weigh compound D-1 (300mg, 0.45mmol) dissolved in 20mL of anhydrous DCM, add TTBP (334.8mg, 1.35mmol, 3eq), diphenyl sulfoxide (109mg, 0.54mmol, 1.2eq), dry 4 angstrom molecular sieves (500 mg), and the solution was stirred at -78°C for 10 minutes. Tf ₂ O (141 mg, 0.5 mmol, 1.1 eq) was added. The solution was stirred for 10 minutes, and benzyl alcohol (54 mg, 0.5 mmol, 1.1 eq) was added and reacted at -78°C for 1 hour. TLC (EA/Hex=1/2) indicated that the conversion of the raw material was complete. Add saturated sodium bicarbonate to adjust the pH to neutral, remove the molecular sieve, concentrate the organic phase under reduced pressure to obtain the crude compound F-1, redissolve it in 20mL of DCM/pyridine (v/v=4/1), add hydrazine acetate (180mg , 1.95mmol, 4.33eq), stirred at room temperature for 2 hours. TLC (EA/Hex=1/2) indicates the disappearance of the raw material. Add acetone to quench the reaction, dilute with dichloromethane, add dilute hydrochloric acid to adjust the pH to neutral, then wash with saturated brine once, dry the organic phase with anhydrous sodium sulfate and filter, concentrate the organic phase, and purify by column chromatography. The eluent ratio (EA/Hex=1/4-1/1) gave compound III-1 (224 mg, 0.41 mmol) as a colorless oil with a yield of 90% (calculated as compound D-1). The NMR data is as follows:

¹ H NMR (600MHz, CDCl ₃ ) δ7.49–7.06 (m, 20H), 5.24 (q, J = 12.3 Hz, 2H), 5.03 (d, J = 1.6 Hz, 1H), 4.74 (d, J = 11.9Hz, 1H), 4.70–4.54 (m, 3H), 4.50 (d, J = 11.9 Hz, 1H), 4.35 (td, J = 9.2, 2.4 Hz, 1H), 4.20 (d, J = 9.3 Hz, 1H), 3.80(dd,J=9.2,3.0Hz,1H), 3.78–3.71(m,1H), 2.82(d,J=2.4Hz,1H).

Example 4: Synthesis of compound IV-1

Weigh compound II-1 (2.0g, 2.84mmol) and compound III-1 (1.7g, 3.12mmol, 1.1eq) and dissolve in 80mL of anhydrous DCM. The solution is reddish brown. Add dry 4 angstrom molecular sieve (1.8 g) After stirring at -40°C for 10 min, TMSOTf (126 mg, 0.57 mmol, 0.2 eq) was added, and the _{reaction was stirred under N 2} protection for 1 hour. TLC (EA/Hex=1/2) indicated that compound II-1 disappeared. Add triethylamine to adjust the pH to neutral, remove the molecular sieve, concentrate the organic phase under reduced pressure, re-dissolve in 20mL of DCM/pyridine (v/v=4/1), add hydrazine acetate (1130mg, 12.3mmol, 4.33eq) , Stir at room temperature for 5 hours. TLC (EA/Hex=1/2) indicates the disappearance of the raw material. Add acetone to quench the reaction, dilute with dichloromethane, add dilute hydrochloric acid to adjust the pH to neutral, then wash with saturated brine once, dry the organic phase with anhydrous sodium sulfate and filter, concentrate the organic phase, and purify by column chromatography. The ratio of the eluent (EA/Hex=1/8-1/1) gave compound IV-1 (1813 mg, 1.8 mmol) as a pale yellow oil with a yield of 64%. The NMR data is as follows:

¹ H NMR(600MHz,CDCl ₃ )δ7.35-6.92(m,35H),5.19(s,1H),5.08(d,J=12.2Hz,1H),5.00(d,J=12.2Hz,1H) , 4.94 (d, J = 12.2Hz, 1H), 4.80 (dd, J = 27.2, 12.1Hz, 2H), 4.71 (d, J = 12.2Hz, 1H), 4.57 (dd, J = 12.1, 4.8 Hz, 2H), 4.52–4.35(m,8H), 4.35–4.28(m,1H), 4.18(t,J=9.5Hz,1H), 4.03-3.93(m,1H), 3.72(t,J=4.2Hz ,1H), 3.64–3.56(m,2H), 3.15(dd,J=9.5,2.8Hz,1H), 2.94(s,1H).

Example 5: Synthesis of β-D-(1,4)-mannuronic acid disaccharide (Compound IX-1)

Compound IV-1 (500mg, 0.5mmol) was dissolved in 22mL of THF/H ₂ O/t-BuOH (v/v/v=1/1/0.2), and palladium on carbon (palladium content 10%) (50mg ), the reaction was stirred at 25°C under hydrogen for 48 hours. TLC (EA/Hex=1/2) indicated that the raw materials disappeared. The palladium-carbon layer was filtered off, and the palladium-carbon layer was washed three times with water (50mL*3). The aqueous phases were combined, and EA( 100 mL) was extracted once, the water was concentrated to 10 mL under reduced pressure, and then freeze-dried to obtain a white solid compound IX-1 (160 mg, 0.4 mmol) with a yield of 86%. The NMR data is as follows:

¹ H NMR(600MHz,D2O)δ5.23-5.12(m,1H), 4.75(d,J=9.4Hz,1H), 4.67-4.57(m,1H), 4.40(t,J=6.8Hz,1H ), 4.08(dt, J=45.1,20.4Hz,1H), 4.02–3.85(m,2H), 3.85–3.67(m,2H), 3.67–3.48(m,1H).

¹³ C NMR (151MHz, D ₂ O) δ 172.85, 172.73, 101.35, 101.06, 99.82, 95.47, 94.08, 93.28, 92.97, 79.52, 77.87, 76.14, 75.36, 75.22, 72.32, 72.27, 71.82, 71.16, 70.11, 69.96, 69.84, 69.80, 69.75, 69.64, 69.59, 69.53, 69.43, 69.21, 69.04, 67.91, 67.79, 67.75, 67.66.

Example 6: Synthesis of compound VI-1

Weigh compound II-1 (2.0g, 2.84mmol) and compound I-1 (1.78g, 3.13mmol, 1.1eq) in 80mL of anhydrous DCM, reddish brown, add dry 4 angstrom molecular sieve (2.0g ), after stirring at -40°C for 10 min, TMSOTf (126 mg, 0.57 mmol, 0.2 eq) was added, and _{stirring under N 2} protection for 1 hour, TLC (EA/Hex = 1/2) indicated that the reaction of compound II-1 was complete. Add saturated sodium bicarbonate to adjust the pH to neutral, filter to remove molecular sieves, extract, dry and concentrate, separate and purify by column chromatography, eluent ratio (EA/Hex=1/8-1/1), to obtain light yellow solid compound VI- 1 (2.7g, 2.42mmol), yield 86%. The NMR data is as follows:

¹ H NMR(600MHz, CDCl ₃ )δ7.39–7.15(m,34H), 6.90(d,J=8.0Hz,2H), 5.68(d,J=6.8Hz,1H), 5.56–5.46(m, 1H), 5.13–5.02(m,2H), 4.92(dd,J=36.4, 12.2Hz, 2H), 4.75–4.53(m,3H), 4.53–4.38(m,7H), 4.38–4.26(m, 1H), 4.16 (s, 1H), 3.83 (dt, J = 9.6, 3.2 Hz, 2H), 3.73 (dd, J = 8.1, 2.7 Hz, 1H), 3.40 (dd, J = 9.6, 2.8 Hz, 1H ), 2.57(dt,J=19.8,7.8Hz,1H),2.52–2.37(m,2H),2.36–2.25(m,1H),2.22(s,3H),2.15–2.05(m,3H)

Example 7: Synthesis of β-D-(1,4)-mannuronic acid tetraose (compound IX-2)

The first step: Weigh VI-1 (500mg, 0.45mmol) and dissolve in 20mL of anhydrous DCM, add TTBP (334.8mg, 1.35mmol, 3eq), diphenyl sulfoxide (109mg, 0.54mmol, 1.2eq) under nitrogen protection , Dry 4 angstrom molecular sieve 500mg), the solution was stirred at -78 ℃ for 10 minutes. Tf ₂ O (141 mg, 0.5 mmol, 1.1 eq) was added. The solution was stirred for 10 minutes, compound IV-1 (500 mg, 0.5 mmol, 1.1 eq) was added, and the reaction was carried out at -78° C. for 1 hour. TLC (EA/Hex = 1/2) indicated that the conversion of the raw material was complete. Add saturated sodium bicarbonate to adjust the pH to neutral, remove the molecular sieve, concentrate the organic phase under reduced pressure, redissolve it in 20mL of DCM/pyridine (v/v=4/1), add hydrazine acetate (180mg, 1.95mmol, 4.33eq ), stirring at room temperature for 2 hours. TLC (EA/Hex=1/2) indicates the disappearance of the raw material. Add acetone to quench the reaction, dilute with dichloromethane, then add dilute hydrochloric acid to adjust the pH to neutral, then wash with saturated brine once, dry the organic phase with anhydrous sodium sulfate and filter, concentrate the organic phase, and purify by column chromatography. The ratio of the eluent (EA/Hex=1/4-1/1) gave compound VIII-1 (766 mg, 0.405 mmol) as a pale yellow solid, with a yield of 90% (calculated as compound VI-1).

Step 2: Dissolve compound VIII-1 (766mg, 0.405mmol) in 22mL of THF/H ₂ O/t-BuOH (v/v/v=1/1/0.2), add palladium on carbon (palladium content 10 %) (300mg), the reaction was stirred at 25°C under hydrogen for 48 hours. TLC (EA/Hex=1/2) indicated that the raw material disappeared. The palladium-carbon layer was filtered off, and the palladium-carbon layer was washed three times with water (50mL*3), and the aqueous phases were combined. , Extracted once with EA (100 mL), concentrated water to 10 mL under reduced pressure, and freeze-dried to obtain white solid compound IX-2 (263 mg, 0.365 mmol) with a yield of 90%.

¹ H NMR(600MHz,D ₂ O)δ5.22(d,J=4.4Hz,1H), 4.98–4.80(m,2H), 4.82–4.76(m,2H), 4.46(d,J=6.6Hz ,1H), 4.17(t,J=6.9Hz,1H), 4.13–3.87(m,8H), 3.87–3.65(m,3H), 3.65–3.48(m,2H).

¹³ C NMR (150MHz, D ₂ O) δ172.68,172.11,171.66,100.40,100.37,99.86,95.47,93.99,92.93,77.94,77.84,77.66,76.54,75.23,74.38,73.03,72.93,72.31,71.81,71.16, 70.97, 69.97, 69.60, 69.53, 69.46, 69.19, 69.04, 67.63.

Example 8: Synthesis of β-D-(1,4)-mannuronic acid triose (compound IX-3)

The first step: Weigh compound II-1 (2.0g, 2.84mmol) and compound IV-1 (3.12g, 3.13mmol, 1.1eq) and dissolve in 100mL of anhydrous DCM, the solution is reddish brown, add dry 4 angstrom molecular sieves (1.8g), stirred at -40 ℃ was added TMSOTf (126mg, 0.57mmol, 0.2eq) after 10min, the reaction was stirred under N ₂ for 1 hour. TLC (EA/Hex=1/2) indicated that compound II-1 disappeared. Add triethylamine to adjust the pH to neutral, remove the molecular sieve, concentrate the organic phase under reduced pressure, re-dissolve in 50mL of DCM/pyridine (v/v=4/1), add hydrazine acetate (1130mg, 12.3mmol, 4.33eq) , Stir at room temperature for 5 hours. TLC (EA/Hex=1/2) indicates the disappearance of the raw material. Add acetone to quench the reaction, dilute with dichloromethane, add dilute hydrochloric acid to adjust the pH to neutral, then wash with saturated brine once, dry the organic phase with anhydrous sodium sulfate and filter, concentrate the organic phase, and purify by column chromatography. The eluent ratio (EA/Hex=1/8-1/1) gave compound V-1 (2547 mg, 1.76 mmol) as a pale yellow oil with a yield of 62%. The NMR data is as follows:

¹ H NMR (600MHz, CDCl3) δ 7.26 (ddd, J = 48.8, 27.9, 19.6 Hz, 50H), 5.25 (s, 1H), 5.14-4.97 (m, 4H), 4.88 (dd, J = 27.9, 12.0Hz, 3H), 4.73 (t, J = 11.9 Hz, 2H), 4.62 (ddd, J = 33.3, 22.0, 11.5 Hz, 6H), 4.54–4.39 (m, 8H), 4.39–4.27 (m, 2H) ), 4.17 (t, J = 8.7 Hz, 1H), 4.04 (s, 1H), 3.79 (d, J = 8.3 Hz, 1H), 3.70 (d, J = 14.7 Hz, 3H), 3.48 (d, J =9.3Hz,1H), 3.45–3.34(m,1H), 3.11(d,J=8.3Hz,1H), 2.82(s,1H).

Step 2: Dissolve compound V-1 (500mg, 0.34mmol) in 20mL of THF/H ₂ O/t-BuOH (v/v/v=1/1/0.2), add palladium on carbon (palladium content 10 %) (50mg), the reaction was stirred at 25°C under hydrogen for 48 hours. TLC (EA/Hex=1/2) indicated that the raw materials disappeared. The palladium-carbon layer was filtered off, and the palladium-carbon layer was washed three times with water (50mL*3), and the aqueous phases were combined. , Extracted once with EA (100 mL), concentrated water to 10 mL under reduced pressure, and freeze-dried to obtain white solid compound IX-3 (167 mg, 0.31 mmol) with a yield of 90%. The NMR data is as follows:

¹ H NMR (600MHz, D ₂ O) δ 5.17 (d, J = 4.3 Hz, 1H), 4.77 (s, 1H), 4.41 (d, J = 6.6 Hz, 1H), 4.11 (d, J = 6.7 Hz,1H),4.05–3.94(m,3H),3.94–3.89(m,2H), 3.88(d,J=9.9Hz,1H), 3.82–3.77(m,1H), 3.77–3.65(m, 3H), 3.58 (dd, J=9.5, 3.0Hz, 1H).

¹³ C NMR (151MHz, D2O) δ185.70,172.66,172.09,171.67,100.34,99.85,93.97,92.93,77.93,77.84,77.73,77.64,74.36,73.03,72.30,71.79,71.12,70.95,70.56,70.08,69.98, 69.55, 69.48, 69.18, 69.02, 67.72, 67.61.

Example 9: Synthesis of β-D-(1,4)-mannuronic acid pentasaccharide (Compound IX-4)

The first step: Weigh VI-1 (500mg, 0.45mmol) and dissolve in 20mL of anhydrous DCM, add TTBP (334.8mg, 1.35mmol, 3eq), diphenyl sulfoxide (109mg, 0.54mmol, 1.2eq) under nitrogen protection , Dry 4 angstrom molecular sieve 500mg), the solution was stirred at -78 ℃ for 10 minutes, Tf ₂ O (141mg, 0.5mmol, 1.1eq) was added, the solution was stirred for 10 minutes, compound V-1 (723mg, 0.5mmol, 1.1 eq), the reaction was carried out at -78°C for 1 hour, and TLC (EA/Hex=1/2) indicated that the conversion of the raw materials was complete. Add saturated sodium bicarbonate to adjust the pH to neutral, remove the molecular sieve, concentrate the organic phase under reduced pressure, redissolve it in 20mL of DCM/pyridine (v/v=4/1), add hydrazine acetate (180mg, 1.95mmol, 4.33eq ), stirring at room temperature for 2 hours. TLC (EA/Hex=1/2) indicates the disappearance of the raw material. Add acetone to quench the reaction, dilute with dichloromethane, add dilute hydrochloric acid to adjust the pH to neutral, then wash with saturated brine once, dry the organic phase with anhydrous sodium sulfate and filter, concentrate the organic phase, and purify by column chromatography. The eluent ratio (EA/Hex = 1/4-1/1) yielded a pale yellow solid compound VIII-2 (935 mg, 0.40 mmol), with a yield of 88% (calculated as compound VI-1).

Step 2: Dissolve compound VIII-2 (935mg, 0.40mmol) in 22mL of THF/H ₂ O/t-BuOH (v/v/v=1/1/0.2), add palladium on carbon (palladium content 10 %) (300mg), the reaction was stirred at 25°C under hydrogen for 48 hours. TLC (EA/Hex=1/2) indicated that the raw material disappeared. The palladium-carbon layer was filtered off, and the palladium-carbon layer was washed three times with water (50mL*3), and the aqueous phases were combined. , Extracted once with EA (100 mL), concentrated water to 10 mL under reduced pressure, and freeze-dried to obtain white solid compound IX-4 (323 mg, 0.36 mmol) with a yield of 90%. The NMR data is as follows:

¹ H NMR (600MHz, D ₂ O) δ 5.17 (s, 1H), 4.86 (s, 1H), 4.63-4.51 (m, 3H), 4.29 (d, J = 5.8 Hz, 1H), 4.05 (s ,1H),3.97(s,3H),3.91(t,J=10.4Hz,2H),3.84(d,J=9.3Hz,6H),3.76(dd,J=14.9,7.5Hz,2H),3.72 (d,J=18.2Hz,3H),3.58(d,J=7.0Hz,1H),3.53(s,1H).

13C NMR (151MHz, D ₂ O) δ 174.35, 174.12, 173.65, 173.37, 172.69, 100.23, 100.15, 99.78, 97.86, 93.81, 93.18, 78.07, 78.00, 77.86, 77.79, 74.50, 72.39, 71.24, 70.38, 70.15, 69.80 ,69.71,69.51,69.01,67.99,67.45,61.48.

Example 10: Synthesis of β-D-(1,4)-mannuronic acid heptaose (compound IX-5)

Step 1: Weigh compound VI-1 (500mg, 0.45mmol) and dissolve it in 20mL of DCM/pyridine (v/v=4/1), add hydrazine acetate (180mg, 1.95mmol, 4.33eq), and stir at room temperature 2 hours. TLC (EA/Hex=1/2) indicates the disappearance of the raw material. Add acetone to quench the reaction, dilute with dichloromethane, add dilute hydrochloric acid to adjust the pH to neutral, then wash with saturated brine once, dry the organic phase with anhydrous sodium sulfate and filter, concentrate the organic phase, and add dry 4 angstroms Molecular sieve (2.0g), re-dissolved in 20mL anhydrous DCM, and then add compound II-1 (317mg, 0.45mmol), the solution is reddish brown, under N ₂ protection, the temperature is controlled below -40 ℃, after stirring for 10 min Add TMSOTf (20mg, 0.09mmol, 0.2eq) and stir for 1 hour. TLC (EA/Hex=1/2) indicated that the reaction of compound II-1 was complete. Add saturated sodium bicarbonate to adjust the pH to neutral, filter to remove molecular sieves, extract, dry and concentrate, mix with silica gel and pass through a column for separation. The ratio of eluent is (EA/Hex=1/8-1/1) to obtain a pale yellow solid compound VII- 1 (560mg, 0.36mmol), yield 80% (based on compound VI-1).

Step 2: Weigh VII-1 (500mg, 0.32mmol) and dissolve in 20mL of anhydrous DCM, add TTBP (238mg, 0.96mmol, 3eq), diphenyl sulfoxide (78mg, 0.38mmol, 1.2eq) under nitrogen protection, Dry 4 angstrom molecular sieve (500mg), the solution was stirred at -78℃ for 10 minutes, Tf ₂ O (100mg, 0.35mmol, 1.1eq) was added, the solution was stirred for 10 minutes, and compound VIII-1 (663mg, 0.35mmol, 1.1 eq), the reaction was carried out at -78°C for 1 hour, and TLC (EA/Hex=1/2) indicated that the conversion of the raw materials was complete. Add saturated sodium bicarbonate to adjust the pH to neutral, remove the molecular sieve, concentrate the organic phase under reduced pressure, redissolve it in 20mL of DCM/pyridine (v/v=4/1), add hydrazine acetate (128mg, 1.39mmol, 4.33eq ), stirring at room temperature for 2 hours. TLC (EA/Hex=1/2) indicates the disappearance of the raw material. Add acetone to quench the reaction, dilute with dichloromethane, add dilute hydrochloric acid to adjust the pH to neutral, then wash with saturated brine once, dry the organic phase with anhydrous sodium sulfate and filter, concentrate the organic phase, and purify by column chromatography. The ratio of the eluent (EA/Hex=1/4-1/1) gave compound VIII-3 (880 mg, 0.27 mmol) as a pale yellow solid, with a yield of 85% (calculated as compound VII-1).

The third step: Dissolve compound VIII-3 (880mg, 0.27mmol) in 22mL of THF/H ₂ O/t-BuOH (v/v/v=1/1/0.2), add palladium on carbon (palladium content 10 %) (300mg), the reaction was stirred at 25°C under hydrogen for 48 hours. TLC (EA/Hex=1/2) indicated that the raw material disappeared. The palladium-carbon layer was filtered off, the palladium-carbon layer was washed three times with water (50mL*3), and the aqueous phases were combined. , Extracted once with EA (100 mL), concentrated water to 10 mL under reduced pressure, and freeze-dried to obtain white solid compound IX-5 (300 mg, 0.24 mmol) with a yield of 89%. The NMR data is as follows:

¹ H NMR (600MHz, D ₂ O) δ 5.96 (d, J = 3.9 Hz, 1H), 5.15 (s, 1H), 4.84 (s, 2H), 4.65 (d, J = 3.9 Hz, 3H), 4.60(s,3H), 4.16(s,1H), 4.10–4.01(m,1H), 3.96(s,3H), 3.91(s,2H), 3.84(d,J=16.4Hz,5H), 3.70 (s, 6H), 3.66 (d, J = 6.7 Hz, 4H), 3.56 (dd, J = 12.2, 6.7 Hz, 3H).

¹³ C NMR (151MHz, D ₂ O) δ174.37,174.05,174.02,174.01,173.94,173.84,173.60,100.23,100.15,99.78,97.86,93.81,93.19,78.08,78.00,77.87,77.79,74.64,72.39,71.25, 70.38, 70.16, 69.80, 69.71, 69.51, 69.01, 68.00, 67.45, 61.49.

Claims (21)

1. A method for preparing β-D-(1,4)-mannuronic acid oligosaccharides represented by formula VIII or formula IX,

   

   include:

   Compound V and compound VII are subjected to a coupling reaction and the protective group R ^{3 is} selectively removed to generate compound VIII;

   

   And optionally, removing the protecting group R ^{2 of} compound VIII at one time to generate β-D-(1,4)-mannuronic acid oligosaccharide as shown in formula IX;

   Wherein, m is selected from an integer of 2-18; n and n'are each independently selected from an integer of 0-8; R ^{1 is} selected from C _1-8 alkyl, optionally substituted _{by C 1-8 alkyl 6-14} aryl group; R ² is a hydroxyl protecting group that can be removed by hydrogenation reaction catalyzed by palladium-carbon or oxidation reaction catalyzed by palladium-carbon ^{; R 3 is hydrogenation reaction catalyzed by palladium-carbon or oxidation reaction catalyzed by palladium-carbon} Removed hydroxyl protecting group.
2. The method according to claim 1, wherein the coupling reaction is carried out in the presence of a large hindered organic base, diphenyl sulfoxide and a sulfonic anhydride catalyst; and then the hydroxyl protecting group R ^{3 at} position 4 and other positions are sequentially removed The hydroxyl protecting group R ² , to obtain the β-D-(1,4)-mannuronic acid oligosaccharide compound shown in IX.
3. The method of claim 2, wherein the sulfonic anhydride catalyst is selected from methanesulfonic anhydride, trifluoromethanesulfonic anhydride, or p-toluenesulfonic anhydride.
4. The method of claim 1, further comprising:

   The coupling reaction of compound II and compound III to generate 1,4-glycosidic bonds, and then selective removal of the protective group R ^{3 of the} 4-hydroxyl group to obtain compound IV;

   

   After that, the compound IV and the compound II are subjected to a coupling reaction to generate 1,4-glycosidic bonds, and then the 4-position hydroxyl protecting group R ^{3 is} selectively removed; optionally, the obtained compound is repeatedly subjected to the above coupling reaction and deprotection Group R ³ until the compound V is obtained;

   

   Wherein, n, R ² and R ^{3 are} as defined in claim 1; R ^{4 is} selected from H, C _1-8 alkyl, C _6-14 aryl; X is selected from fluorine, chlorine, bromine, and iodine.
5. The method according to claim 4, wherein the coupling reaction is carried out in the presence of a sulfonic acid catalyst; the sulfonic acid catalyst is selected from the group consisting of methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid or trifluoromethanesulfonic acid Trimethylsilyl acid.
6. The method of claim 1, further comprising:

   The coupling reaction of compound I and compound II to generate 1,4-glycosidic bonds, and then selective removal of the protective group R ^{3 of the} 4-hydroxyl group to obtain compound VI;

   

   The compound VI and compound II are subjected to a coupling reaction to generate 1,4-glycosidic bonds, and then the 4-position hydroxyl protecting group R ^{3 is} selectively removed; optionally, the obtained compound is continued to repeat the above coupling reaction and deprotection Group R ³ until the compound VII is obtained;

   

   Wherein, n', R ¹ , R ² and R ^{3 are} as defined in claim 1; R ^{4 is} selected from H, C _1-8 alkyl, C _6-14 aryl; X is selected from fluorine, chlorine, bromine, iodine.
7. The method of claim 6, wherein the coupling reaction is carried out in the presence of a sulfonic acid catalyst; the sulfonic acid catalyst is selected from the group consisting of methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid or trifluoromethanesulfonic acid Trimethylsilyl acid.
8. The method of any one of claims 4-7, wherein compound II is obtained as follows:

   

   Step 1: Protect the 4-hydroxyl of compound I to obtain compound D;

   Step 2: Selectively remove the anomeric carbon protecting group -SR ^{1 of} compound D to obtain compound E;

   Step 3: Under alkaline conditions, compound E is reacted with CX ₃ C(=NR ⁴ )X or CX ₃ CN to obtain compound II;

   Wherein R ¹ , R ² and R ^{3 are} as defined in claim 1; R ^{4 is} selected from H, C _1-8 alkyl, C _6-14 aryl; X is selected from fluorine, chlorine, bromine, and iodine.
9. The method of any one of claims 4-5, wherein compound III can be obtained as follows:

   

   Step 1: Protect the 4-hydroxyl of compound I to obtain compound D;

   Step 2: Under alkaline conditions, ^{react compound D with R 2} OH to obtain compound F;

   ^{Step 3: Selectively remove the protective group R 3} of the 4-hydroxyl group of compound F to obtain compound III;

   Wherein R ¹ , R ² and R ^{3 are} as defined in claim 1.
10. The method according to any one of claims 6-9, further comprising:

    

    Step 1: 1,2,3,4,6-Penta-O-acetyl-D-mannanose ^{reacts with the anomeric carbon protecting agent R 1} SH, and then undergoes a hydrolysis reaction to obtain compound A;

    Step 2: Selectively protect the 2 and 3 hydroxyl groups of compound A to obtain compound B;

    Step 3: Oxidize the hydroxyl group at the 6-position of compound B to a carboxyl group, and then esterify the carboxyl group to obtain compound I;

    Wherein R ¹ and R ^{2 are} as defined in claim 1.
11. The method of any one of claims 1-10, wherein:

    R ^{2 is} selected from C _6-14 arylmethyl or allyl, and the C _6-14 arylmethyl is optionally substituted by C _1-8 alkyl, C _1-8 alkoxy, or halogen;

    R ^{3 is} selected from C _1-8 alkyl acyl, C _1-8 alkoxy acyl, C _6-14 aryl acyl, tris (C _1-8 alkyl) silyl, 9-fluorenyl methoxymethyl Acyl, tris (C _6-14 aryl) methyl; wherein any carbon atom in the C _1-8 alkyl group in the _{C 1-8} alkyl acyl group and the C _{1-8 alkoxy acyl group can be optionally} Oxo.
12. The method of claim 11, wherein:

    R ^{2 is} selected from benzyl, p-methoxybenzyl, naphthylmethyl, and allyl;

    R ^{3 is} selected from acetyl, levulinyl, trimethylsilyl, tert-butyldimethylsilyl, benzoyl, 9-fluorenylmethoxyformyl, trityl.
13. Compound of formula I:

    

    Or its salt, in which,

    R ^{1 is} selected from C _1-8 alkyl, C _6-14 aryl _{optionally substituted by C 1-8 alkyl;}

    R ^{2 is} selected from a C _6-14 arylmethyl group or an allyl group, and the C _6-14 arylmethyl group is optionally substituted by a C _1-8 alkyl group, a C _1-8 alkoxy group, or halogen.
14. The compound of formula I or its salt according to claim 13, wherein:

    R ^{1 is} selected from phenyl, o-tolyl, p-methylphenyl, 4-tert-butyl-2-methylphenyl, 2,4-di-tert-butylphenyl, methyl or ethyl;

    R ^{2 is} selected from benzyl, p-methoxybenzyl, naphthylmethyl, and allyl.
15. The preparation method of the compound of formula I or its salt according to claim 13 or 14, comprising:

    

    Step 1: 1,2,3,4,6-Penta-O-acetyl-D-mannanose ^{reacts with the anomeric carbon protecting agent R 1} SH, and then performs an alkaline hydrolysis reaction to obtain compound A;

    Step 2: Selectively protect the 2 and 3 hydroxyl groups of compound A to obtain compound B;

    Step 3: Oxidize the hydroxyl group at the 6-position of compound B to a carboxyl group, and then esterify the carboxyl group to obtain compound I;

    Wherein R ¹ and R ^{2 are} as defined in claim 13 or 14.
16. Compound of formula II:

    

    Or its salt, in which,

    R ^{2 is} selected from C _6-14 arylmethyl or allyl, and the C _6-14 arylmethyl is optionally substituted by C _1-8 alkyl, C _1-8 alkoxy, or halogen;

    R ^{3 is} selected from C _1-8 alkyl acyl, C _1-8 alkoxy acyl, C _6-14 aryl acyl, tris (C _1-8 alkyl) silyl, 9-fluorenyl methoxymethyl Acyl, tris (C _6-14 aryl) methyl; wherein any carbon atom in the C _1-8 alkyl group in the _{C 1-8} alkyl acyl group and the C _{1-8 alkoxy acyl group can be optionally} Oxo

    R ^{4 is} selected from hydrogen, C _1-8 alkyl, C _6-14 aryl;

    X is selected from fluorine, chlorine, bromine, and iodine.
17. The compound of formula II or its salt according to claim 16, wherein:

    R ^{2 is} selected from benzyl, p-methoxybenzyl, naphthylmethyl, and allyl;

    R ^{3 is} selected from acetyl, levulinyl, trimethylsilyl, tert-butyldimethylsilyl, benzoyl, 9-fluorenylmethoxyformyl, trityl;

    R ^{4 is} selected from hydrogen, C _1-4 alkyl, C _6-8 aryl

    X is selected from chlorine or bromine.
18. The preparation method of the compound of formula II or its salt according to claim 16 or 17, comprising:

    

    Step 1: Protect the 4-hydroxyl of compound I to obtain compound D;

    Step 2: Selectively remove the anomeric carbon protecting group -SR ^{1 of} compound D to obtain compound E;

    Step 3: Under basic conditions, compound E is reacted with tri-CX ₃ C(=NR ⁴ )X or CX ₃ CN nitrile to obtain compound II;

    Wherein R ^{1 is} as defined in claim 1; R ² , R ³ , R ⁴ and X are as defined in claim 16 or 17.
19. Compound of formula III:

    

    Or its salt, in which,

    R ^{2 is} selected from a C _6-14 arylmethyl group or an allyl group, and the C _6-14 arylmethyl group is optionally substituted by a C _1-8 alkyl group, a C _1-8 alkoxy group, or halogen.
20. The compound of formula III or its salt according to claim 19, wherein:

    R ^{2 is} selected from benzyl, p-methoxybenzyl, naphthylmethyl, and allyl.
21. The preparation method of the compound of formula III or its salt according to claim 19 or 20, comprising:

    

    Step 1: Protect the 4-hydroxyl of compound I to obtain compound D;

    Step 2: Under alkaline conditions, ^{react compound D with R 2} OH to obtain compound F;

    ^{Step 3: Selectively remove the protective group R 3} of the 4-hydroxyl group of compound F to obtain compound III;

    Wherein R ¹ and R ^{3 are} as defined in claim 1, and R ^{2 is} as defined in claim 19 or 20.

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